The inventor of the present invention has found that it becomes possible to copy a gene (target gene), which encodes a target protein, in an intercellular copy number increased to approximately 100,000 copies simply by plasmid conduction into human-derived cancer cells (COLO 320 colon cancer cell line, and HeLa cell line) by lipofection, the plasmid (hereinafter, “IR/MAR plasmid”) having mammalian copying initiation region (IR; initiation region) and a matrix attachment region (MAR; matrix attachment region), and selecting by utilizing a gene tolerance to a chemical (Blasticidin or Neomycin), and that the mass amplification of the target gene can be attained regardless of whether the target gene has a gene structure identical to that of the IR/MAR plasmid (i.e., the target gene has a cis gene structure) or the target gene has a gene structure different from that of the IR/MAR plasmid (i.e., the target gene has a trans gene structure) (see Patent Document 1, Patent Document 2, Non-Patent Document 1, and Non-Patent Document 2).
A cell line in which an IR/MAR plasmid and a target gene were transfected, was analyzed quantitatively as to a transcription amount of mRNA from the target gene. This analysis found that the transcription amount of mRNA was not increased while the copy number of the target gene was increased. It was deduced that the transcription was repressed due to a repeated sequence produced by the mass amplification of a region including the target gene.
One known method of releasing the transcription repression caused by the repeated gene sequence is, for example, treating the cells with histone acetylating enzyme inhibitor such as trichostatin A (see Non-Patent Document 3).
[Patent Document 1]
    Japanese Unexamined Patent Application Publication, Tokukai, No. 2003-245083 (published on Sep. 2, 2003)[Patent Document 2]    Japanese Unexamined Patent Application Publication, Tokukai, No. 2004-337066 (published on Dec. 2, 2004)[Non-Patent Document 1]    Noriaki Shimizu, et al. (2001) Plasmids with a Mammalian Replication Origin and a Matrix Attachment Region Initiate the Event Similar to Gene Amplification. Cancer Research vol. 61, no. 19, p 6987-6990.[Non-Patent Document 2]    Noriaki Shimizu, et al (2003) Amplification of plasmids containing a mammalian replication initiation region is mediated by controllable conflict between replication and transcription. Cancer Research, vol. 63, no. 17, p 5281-5290.[Non-Patent Document 3]    McBurney, M. W. et al, Exp Cell Res (2002), vol 274, p 1-8
Therefore, there is such a problem in that even if a polynucleotide containing the amplified target gene is amplified in order to mass-produce a protein via the amplification of the target gene, the transcription of the target gene is repressed once the repeated sequence is created thereby failing to express the protein of the final target.
Sole application of the method of treating the cells with histone acetylating enzyme inhibitor such as trichostatin A as disclosed in Non-Patent Document 3 could not sufficiently release the transcription repression caused by the repeated sequence of the gene amplified to several thousand to approximately 10 thousand copies.
An object of the present invention is to provide a method and kit etc. for releasing the transcription repression caused by the repeated sequence of the gene, and to establish a system for mass production of useful protein by gene amplification.